PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1–4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5–9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2–EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.


Article
mice compared with C57BL/6 wild-type (WT) mice revealed normal CD4 + and CD8 + T cell abundance and subset composition in lymphoid organs (Extended Data Fig. 1a-g).Unaltered T cell composition was similarly observed in Gzmb cre Ptger2 −/− Ptger4 fl/fl mice, which lack EP 2 and EP 4 in CD8 + T cells expressing granzyme B (GZMB) (Extended Data Fig. 1a-g).Notably, after tumour challenge, Cd4 cre Ptger2 −/− Ptger4 fl/fl mice exhibited improved tumour immune control, and fully rejected tumours formed by immune-evasive, PGE 2 -producing (control) BRAF V600E melanoma cells (Fig. 1a and Extended Data Fig. 2a).This was not the case for Ptger2 −/− Ptger4 f l/fl mice and WT mice, in which control BRAF V600E tumours progressively grew (Fig. 1a and Extended Data Fig. 2a).We further validated that BRAF V600E melanoma depended on tumour-derived PGE 2 to evade anticancer immunity by demonstrating that COX-deficient Ptgs1/Ptgs2 −/− BRAF V600E melanoma, which lacks PGE 2 production, failed to escape immune control (Fig. 1a and Extended Data Fig. 2a).We also confirmed that this effect is mediated by CD8 + T cells 14 (Extended Data Fig. 2b).Extending our analysis to other mouse tumour models, tumours formed by Panc02 pancreatic cancer cells similarly exhibited complete regression in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice but not in Ptger2 −/− Ptger4 fl/fl or WT mice (Fig. 1b and Extended Data Fig. 2c).Similar results were observed for tumours derived from MC38 colorectal cancer cells (Extended Data Fig. 2d,e).
Immune control of control BRAF V600E melanoma tumours in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice was linked to markedly increased CD8 + TIL accumulation (Fig. 1c-e).By contrast, no substantial differences were observed for CD4 + TILs (Fig. 1c-e).This result suggests that although PGE 2 -EP 2 /EP 4 signalling may affect CD4 + T cell function, these cells, at least in BRAF V600E tumours, do not have a major role in immune escape.Consistently, antibody-mediated T cell depletion confirmed the relevance of CD8 + but not CD4 + T cells for immune control of PGE 2 -producing BRAF V600E tumours in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice (Fig. 1f).Taken together, these data suggest that EP 2 /EP 4 signalling controls the accumulation of CD8 + TILs in PGE 2 -producing tumours and that this is important for cancer immune evasion.

PGE 2 does not affect CD8 + T cell priming
Priming of anticancer CD8 + T cells in tumour-draining lymph nodes (tdLNs) by type 1 conventional dendritic cells (cDC1s) that transport tumour antigens to tdLNs is thought to underlie anticancer CD8 + T cell responses 21,22 .To test whether PGE 2 impairs cDC1-mediated CD8 + T cell priming, we injected WT mice with PGE 2 -producing control or PGE 2 -deficient Ptgs1/Ptgs2 −/− BRAF V600E melanoma cells engineered to express the model antigen ovalbumin (OVA).We then determined the presence of migratory CD103 + cDC1 cross-presenting OVA-derived peptides on major histocompatibility complex (MHC) class I molecules in tdLNs (Extended Data Fig. 3a).Migratory cDC1s in both models cross-presented tumour-derived OVA protein with similar efficiency, as determined by staining for OVA(257-264) (SIINFEKL) peptide loading of the MHC class I molecule H-2K b (Extended Data Fig. 3b,c).To further examine T cell priming, we adoptively transferred naive CD8 + OT-I T cells (Extended Data Fig. 3d), which express a transgenic T cell receptor (TCR) specific for OVA, into mice subsequently transplanted with BRAF V600E -OVA tumours.Naive (CD44 low ) OT-I T cells efficiently expanded into CD44 + TCF1 + OT-I T cells within tdLNs in both groups (Extended Data Fig. 3e,f).This result demonstrates that T cell priming is unaffected.Consistent with these data, we did not detect substantial PGE 2 levels in tdLNs from control BRAF V600E tumours or in other distant organs (Extended Data Fig. 3g).Moreover, progressive outgrowth of control BRAF V600E tumours and efficient immune control of Ptgs1/Ptgs2 −/− BRAF V600E tumours was unchanged following tumour transplantation to the same lymph drainage site (Extended Data Fig. 3h).These findings imply that an anticancer CD8 + T cell response initiated in the shared tdLN achieves effective elimination of the PGE 2 -deficient tumour but nevertheless fails in the co-transplanted PGE 2 -producing tumour.Taken together, these data demonstrate that PGE 2 controls anticancer CD8 + T cell responses locally within tumour tissue, which raises the question of how it affects CD8 + TILs.

PGE 2 controls CD8 + TIL effector expansion
CD8 + TILs are heterogenous and comprise at least two phenotypically and functionally distinct populations: (1) proliferation and differentiation competent TCF1 + cells that lack cytotoxic effector functions (often referred to as 'stem-like' or 'precursor of exhausted' T cells); and (2) TIM-3 + (TCF1 − ) cells that encompass more differentiated effector and terminally differentiated or exhausted T cells.TCF1 + CD8 + T cells fulfil an essential role in anticancer immunity by giving rise to TIM-3 + progeny through proliferative expansion and effector differentiation 2,5,8,9 .This process is pivotal for anticancer immunity that at least in part occurs locally within tumour tissue [1][2][3] .
Our results raised the question of whether interference with effector differentiation of TCF1 + CD8 + TILs underlies the PGE 2 -mediated impairment of anticancer immunity.To address this issue across the single-cell landscape of CD8 + TILs, we performed parallel single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) of CD8 + TILs sorted from BRAF V600E tumours at day 11 after tumour transplantation into Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Ptger2 −/− Ptger4 fl/fl mice (as control) (Fig. 2a and Extended Data Fig. 4a).We also included Gzmb cre Ptger2 −/− Ptger4 fl/fl mice (Fig. 2a), reasoning that this would enable us to determine the impact of EP 2 /EP 4 -mediated PGE 2 signalling on those CD8 + T cells undergoing effector differentiation within tumour tissue.We further included four biological replicates in each group to ensure that heterogeneity among individual tumours is reflected in our analysis.scRNA-seq analysis revealed eight TIL clusters (Fig. 2b) that all expressed Pdcd1 (which encodes PD-1), the activation marker Cd44 and the transcription factor (TF) Tox (Extended Data Fig. 4b), a result consistent with their activation history.Of note, CD8 + TILs displayed equally high protein expression of CD44, TOX and PD-1 that did not differ among cells isolated from tumours in Ptger2 −/− Ptger4 fl/fl mice, Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice (Extended Data Fig. 4c).
In our concatenated scRNA-seq data, TIL clusters 1 and 2 shared high expression of stem-like T cell markers such as Tcf7 (which encodes TCF1), Slamf6 and Il7r (Fig. 2c,d and Extended Data Fig. 4d).Of note, both of these clusters displayed markedly higher expression of gene signatures of memory or tumour-reactive T cells than signatures for naive T cells (Extended Data Fig. 4e,f), which indicated that at least a substantial fraction of these cells is antigen-experienced.TCF1 + TILs in cluster 1 displayed enriched expression of Sell (which encodes CD62L), Ccr7 and Bach2 (Extended Data Fig. 4d,g,h).By contrast, TCF1 + TILs in cluster 2 lacked Sell but showed expression of markers associated with effector function (such as Gzmb, Gzmk and Fasl) and migration (S1pr1, Itga4, Gpr183, Itgb1, Cxcr3 and Ier2) (Fig. 2d and Extended Data Fig. 4d,g,h), which indicated their incipient effector differentiation.
Consistently, CD62L − TCF1 + TILs but not CD62L + TCF1 + TILs stained positive for intracellular GZMB protein (Extended Data Fig. 4i), although GZMB expression in these cells was low both in terms of frequency of GZMB + cells and total GZMB levels.The remaining scRNA-seq clusters (clusters 3-8) lacked Tcf7 expression and, in addition to Gzmb, shared expression of genes associated with T cell differentiation and effector function, including Havcr2 (which encodes TIM-3) and high expression of the chemokine receptor Cxcr6 (Fig. 2c,d and Extended Data Fig. 4g,h), which identified them as more differentiated early and/or terminally differentiated TIL populations.We confirmed co-expression of TIM-3 and CXCR6 at the protein level (Extended Data Fig. 4j) and used both molecules as overarching markers to collectively denote (TCF1 − ) effector TILs.Among the different clusters of TIM-3 + CXCR6 + TILs, clusters 3 and 4 were marked by high expression of molecules associated with early effector-like cells; for example, Cx3cr1 (refs.23,24)  in cluster 3 and Cd7 (ref.25) in cluster 4 (Extended Data Fig. 4d,h).By contrast, clusters 5-8 displayed increased expression of cytotoxic effector molecules and immune-inhibitory receptors (Fig. 2d and Extended Data Fig. 4h), but were distinguished by differential expression of cytokines (for example, Ifng and Tnf), molecules associated with growth arrest and DNA repair (Apex1 and Gadd45b) and type I interferon signalling (Isg15, Ifit1 and Ifit3) (Extended Data Fig. 4d,h).Notably, in contrast to tumour tissue, we did not detect any GZMB + cells among activated CD44 + CD8 + T cells in tdLNs or spleen of tumour-bearing mice (Extended Data Fig. 4k).This result supports the notion that effector differentiation of anticancer CD8 + T cells occurs within tumour tissue.Unsupervised slingshot analysis of our TIL scRNA-seq data uncovered a tree-shaped developmental trajectory that begins with TCF1 + cells and progresses over CX 3 CR1 hi TIM-3 + effector cells into CD7 hi TIM-3 + effector cells, from which it branches off into distinct terminally differentiated T cell populations (Fig. 2e).Together, these results indicate a progressive trajectory for TIL differentiation within tumours that originates from TCF1 + TILs and follows a unidirectional path of effector differentiation before ending in multiple smaller branches of terminally differentiated TIL populations.
To assess the impact of PGE 2 -EP 2 /EP 4 signalling on the landscape of CD8 + TILs, we separated our scRNA-seq data on the basis of recipient mouse groups.Density analysis revealed a prominent shift towards early effector (clusters 3 and 4) and terminally differentiated TIL populations (cluster 5) in both Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice compared with Ptger2 −/− Ptger4 fl/fl mice (Fig. 2f).We therefore quantified the frequencies TIL populations across all replicates.Ptger2 −/− Ptger4 fl/fl mice lacked expansion of any differentiating effector TIL populations (Fig. 2g).By contrast, in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice, we detected elevated frequencies of early and late effector TIL populations that further increased progressively along the common trajectory of effector differentiation (clusters 2-5; Fig. 2g).This pattern was similarly observed for Gzmb cre Ptger2 −/− Ptger4 fl/fl mice (Fig. 2g), in which TIL expansion was even more prominent, which probably reflects additional favourable activity of intratumoural GZMB + natural killer cells 15 .Enhanced TIL expansion in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice correlated with the fact that TCF1 + and TIM-3 + CXCR6 + TILs in both models had lost Ptger2 and Ptger4 expression (Extended Data Fig. 5a-d).Consistent with the notion that intratumoural effector differentiation causes the loss of EP 4 in TCF1 + TILs in Gzmb cre Ptger2 −/− Ptger4 fl/fl mice, Gzmb cre Ptger2 −/− Ptger4 fl/f TCF1 + CD8 + T cells generated in vitro displayed efficient Ptger4 ablation in an effector differentiation assay (Extended Data Fig. 5e,f).In line with enhanced TIL expansion, expression of a proliferation signature in effector TIL populations from Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice was higher than from Ptger2 −/− Ptger4 fl/fl mice (Extended Data Fig. 5g).Consistently, EP 2 /EP 4 -deficient TCF1 + TILs and their TIM-3 + progeny displayed increased expression of the proliferation marker Ki-67 (Extended Data Fig. 5h).However, we did not detect a substantial gain in expression of a gene signature for cytotoxic Article effector function in these TIL populations (Extended Data Fig. 5i).Thus, PGE 2 does not modify the expression of genes associated with T cell function but prevents their differentiation and expansion, which highlights a mechanistic difference to canonical factors that drive dysfunctional CD8 + T cell responses through transcriptional programming, such as TOX 26,27 or MYB 28 .Taken together, these findings suggest that tumour-derived PGE 2 locally impairs the differentiation and expansion of effector T cell populations arising from TCF1 + TILs.Moreover, EP 2 /EP 4 deficiency rescues TILs from this inhibitory effect of PGE 2 .

TCF1 + TIL effector expansion achieves tumour control
We next sought to provide further evidence that EP 2 /EP 4 deficiency in CD8 + T cells permits productive effector differentiation of TILs in PGE 2 -producing tumours.Quantification of TIL populations across PGE 2 -deficient Ptgs1/Ptgs2 −/− BRAF V600E tumours from WT mice and PGE 2 -producing control BRAF V600E from WT mice, Ptger2 −/− Ptger4 fl/fl mice and Cd4 cre Ptger2 −/− Ptger4 fl/fl mice (Extended Data Fig. 7a-c) revealed that the numbers of TCF1 + TILs were comparable among all groups (Extended Data Fig. 7b).This result indicated that the generation of TCF1 + CD8 + T cells in lymphoid tissues and their tumour infiltration is not affected by PGE 2 .However, whereas the numbers of differentiated TIM-3 + TILs were low in control BRAF V600E tumours in WT mice and Ptger2 −/− Ptger4 fl/fl mice, they were highly abundant in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice (Extended Data Fig. 7c) and indistinguishable from those found in Ptgs1/Ptgs2 −/− BRAF V600E tumours in WT mice.
To determine whether TIM-3 + TILs were generated from TCF1 + TILs locally within tumour tissue, we made use of our finding that at early stages after implantation (day 6), tumours contain TCF1 + TILs but not (yet) differentiated TIM-3 + TILs (Fig. 2j-n).Tumour-bearing Cd4 cre Ptger2 −/− Ptger4 fl/fl mice treated from day 6 onwards with the S1P1R antagonist FTY720, which prevents lymph node (LN) egress of newly primed CD8 + T cells 31 , showed unabated intratumoural development and prominent expansion of TIM-3 + TILs over time (Fig. 2k-m).By contrast, when initial tumour infiltration of TCF1 + CD8 + T cells was blocked by FTY720 application from day 1 onwards, no intratumoural TIL expansion was detected (Fig. 2m).Notably, the proliferative response originating from TCF1 + TILs present in tumour tissue at day 6 was sufficient to achieve control of tumour growth (Fig. 2n).This result demonstrates that TCF1 + TILs locally generate potent anticancer effector responses when protected from inhibitory PGE 2 signalling in tumours.
We therefore tested whether PGE 2 controls the IL-2-mediated expansion of TCF1 + TILs sorted from PGE 2 -deficient Ptgs1/Ptgs2 −/− tumours (identified as TIM-3 − CXCR6 − TILs; Extended Data Fig. 8e).PGE 2 strongly compromised the capacity of TCF1 + TILs to expand and differentiate into effector cells when stimulated with high-dose IL-2 together with anti-CD3 and anti-CD28 (anti-CD3/CD28) treatment 35,36 (Fig. 3b).Bypassing the scarcity of TIL numbers, we further addressed this issue using antigen-experienced, repetitively activated TCF1 + CD8 + T cells generated in vitro, on which PGE 2 had an identical inhibitory effect (Fig. 3c).In line with PGE 2 -mediated impairment of IL-2-driven proliferation and effector differentiation, PGE 2 -treated TCF1 + CD8 + T cells from in vitro T cell cultures showed markedly reduced DNA replication early after their stimulation (Fig 3d).Transcriptional profiling by RNA-seq (Fig. 3e) revealed that PGE 2 exposure resulted in distinct transcriptional changes in stimulated TCF1 + CD8 + T cells and their unstimulated counterparts (Fig. 3f).Analyses of the stimulated T cell populations identified 294 differentially expressed genes (DEGs) following PGE 2 exposure (Fig 3g).PGE 2 -treated TCF1 + CD8 + T cell populations expressed increased levels of transcripts encoding for molecules related to EP 2 /EP 4 -mediated cAMP signalling (Crem and Fosl2) and T cell quiescence (for example, Phlpp1, Klf3 and Klf4) (Fig. 3g and Extended Data Fig. 8f).Gene set enrichment analysis (GSEA) showed a selective downregulation of the T cell differentiation-associated mTORC1 signalling pathway and the IL-2 signalling pathway (Fig. 3h).The latter result is consistent with the observed reduced IL-2 pathway activity in TILs identified in our scRNA-seq analysis and further supports the notion that PGE 2 impairs the proliferative expansion of TILs through the inhibition of IL-2 signalling.
In line with an inhibitory effect of PGE 2 on IL-2 signalling, IL-2 stimulation failed to promote STAT5 phosphorylation (pSTAT5) in PGE 2 -treated TCF1 + CD8 + T cells (Fig. 3i).Notably, this defect was associated with reduced surface expression of the IL-2R gamma chain (IL-2Rγc) (Extended Data Fig. 8g) and could only partially be rescued by stimulation with high doses of IL-2 (Fig. 3j).This result points towards a dominant inhibitory effect of PGE 2 on IL-2 signalling through the regulation of IL-2Rγc expression.Consistent with this notion, PGE 2 impaired the expansion of TCF1 + CD8 + T cells not only in response to IL-2 but also the γc cytokines IL-7 and IL-15 (Fig. 3k).Thus, PGE 2 fundamentally affects the entire γc cytokine signalling pathway in TCF1 + CD8 + T cells and their differentiating progeny.IL-2-mediated pSTAT5 (Fig. 3l,m) and IL-2-dependent T cell proliferation and expansion (Fig. 3n and Extended Data Fig. 8h,i) was rescued in TCF1 + CD8 + T cells from Cd4 cre Ptger2 −/− Ptger4 fl/fl mice, which demonstrates the functional relevance of EP 2 /EP 4 signalling for the restriction of IL-2 signalling.Taken together, these data suggest that the PGE 2 -EP 2 /EP 4 axis limits productive anticancer TIL responses by suppressing the IL-2 signalling pathway.

EP 2 /EP 4 -deficient TILs mediate cancer elimination
To examine antigen-specific TIL responses in more detail, we used WT (EP 2 /EP 4 -proficient) and Cd4 cre Ptger2 −/− Ptger4 fl/fl (EP 2 /EP 4 -deficient) OT-I T cells.We co-transferred a small number (1 × 10 3 cells) of congenically marked WT and EP 2 /EP 4 -deficient OT-I T cells into recipient mice, which were subsequently challenged with MC38-OVA tumours (Fig. 4a).Consistent with the observation that PGE 2 selectively inhibits CD8 + T cells within tumours, both WT and EP 2 /EP 4 -deficient OT-I T cells displayed prominent and unrestricted expansion in tdLNs (Fig. 4b,c).However, whereas the response by WT OT-I T cells collapsed after the expand in tumours (Extended Data Fig. 9g,h) and were able to give rise to TIM-3 + CXCR6 + TILs (Extended Data Fig. 9i).In separate experiments, we also observed that the development of TIM-3 + effector progeny from TCF1 + EP 2 /EP 4 -deficient OT-I T cells exclusively occurred in tumours but not in tdLNs (Fig. 4e,f and Extended Data Fig. 9j).Together, these data suggest that clonal T cell effector differentiation is restricted to tumour tissue and originates from TCF1 + TILs 1-3 .Consistent with this notion, and similar to our observations for polyclonal anticancer CD8 + T cell responses, FTY720-mediated blockade of T cell egress

Article
from LNs from day 6 onwards had no impact on the local expansion of EP 2 /EP 4 -deficient OT-I TILs (Extended Data Fig. 9k,l).We conclude that tumour-specific TCF1 + TILs expand and give rise to effector progeny within tumours, and this pivotal phase of the anticancer CD8 + T cell responses is blunted by PGE 2 .
Finally, to specifically evaluate whether EP 2 /EP 4 -deficient antigenspecific CD8 + T cells mount protective anticancer responses, we analysed the growth of MC38-OVA tumours transplanted into WT mice with or without transfer of WT or EP 2 /EP 4 -deficient OT-I T cells.EP 2 /EP 4 -deficient OT-I T cells achieved complete rejection of MC38-OVA tumours, whereas WT OT-I T cells failed to affect progressive MC38-OVA tumour growth (Fig. 4n).Of note, EP 2 /EP 4 -deficient OT-I but not WT OT-I TILs also showed enhanced expansion that led to efficient tumour elimination in mouse melanoma D4M.3A-pOVA tumours (Extended Data Fig. 9m,n).Taken together, these results suggest that interfering with the PGE 2 -EP 2 /EP 4 axis in cancer-specific CD8 + T cells can elicit their expansion and effector differentiation within tumours and result in protective T cell-mediated anticancer immunity.

Discussion
Our results demonstrate that tumour-derived PGE 2 acts locally within the tumour microenvironment to limit CD8 + TIL expansion and effector differentiation originating from TCF1 + stem-like TILs.This inhibitory mechanism is crucial for cancer immune escape.We reveal that PGE 2 -mediated restriction of TIL responses generated from TCF1 + TILs depends on TIL-intrinsic signalling of the PGE 2 -receptors EP 2 and EP 4 , which causes downregulation of functional IL-2 receptors and curtails TIL responsiveness to IL-2.As a result, interference with PGE 2 -EP 2 /EP 4 signalling in CD8 + T cells enhances their IL-2 responsiveness and induces protective TIL-mediated anticancer immunity.Of note, the effect of PGE 2 on TIL expansion and effector differentiation may at least in part be linked to a defect in IL-2-dependent mTORC1 signalling, as also suggested by an accompanying paper 37 .
Beyond highlighting that clonal expansion and effector differentiation of stem-like TCF1 + CD8 + T cells occurs within tumour tissue, as recently suggested [1][2][3] , our results reveal that this critical phase of protective anticancer immunity is selectively targeted by tumour-derived PGE 2 .These findings therefore identify an intratumoural checkpoint that locally controls expansion and effector differentiation of cancer-specific CD8 + TILs.Of note, this mechanism may act in parallel to PGE 2 -mediated inhibition of cDC1 (ref.38), which can support TCF1 + TIL responses within the tumour microenvironment 39 .
Our unbiased transcriptional profiling by scRNA-seq uncovered that protective anticancer responses by EP 2 /EP 4 -deficient TILs are coupled to a rescue of IL-2 signalling.Recent studies have highlighted the relevance of IL-2 signalling for the generation of effective CD8 + T cell responses from antigen-specific TCF1 + CD8 + T cells 6,34,40,41 .Therefore, the discovery that the PGE 2 -EP 2 /EP 4 axis antagonizes the responsiveness of TCF1 + TILs to IL-2 has important mechanistic and clinical implications.Our results provide evidence that PGE 2 limits the proliferative capacity (and hence likely the self-renewal) of TCF1 + stem-like TILs and at the same time curbs effector T cell generation along the entire pathway of intratumoural TIL differentiation.Importantly, ablation of PGE 2 signalling and consequently reconstitution of IL-2 signalling sufficed to achieve clonal TIL expansion and their effector differentiation within tumours that was not accompanied while preserving TCF1 + stem-like TILs.This is fundamentally different to interfering with exhaustion-inducing transcription factors such as TOX or MYB, which comes at the expense of TCF1 + stem-like CD8 + T cells and leads to a substantial change towards the development of terminally differentiated dysfunctional T cells [26][27][28] .Moreover, our finding that abrogating PGE 2 signalling in T cells enhances clonal expansion across the entire differentiation spectrum of anticancer TILs indicates that physiological IL-2 concentrations within tumours are sufficient to drive protective anticancer immunity if IL-2 signalling in TILs is restored.
Targeting EP 2 and EP 4 on anticancer T cells to overcome PGE 2 -induced curtailing of IL-2 responsiveness might be preferential over using high concentrations of IL-2, as the latter may lead to deleterious off-target effects of IL-2 on IL-2R-expressing lung endothelial cells or CD4 + regulatory T cells 42 .On this note, ablation of the PGE 2 -EP 2 /EP 4 signalling axis to enhance IL-2 responsiveness in adoptively transferred cancer-specific CD8 + T cells bears the promise to unleash their full potential to mount protective anticancer immunity not only in mice but also in cancer patient-derived TILs, as demonstrated in the accompanying paper 37 .Given the association of increased COX-mediated PGE 2 -production in tumours with cancer growth and poor survival rates in patients with cancer, our findings therefore identify the PGE 2 -EP 2 /EP 4 signalling axis in TILs as molecular target to improve T cell immune therapy in cancer patients with PGE 2 -producing tumours.This strategy may further be beneficial in tumours that produce high levels of other EP 2 /EP 4 -engaging prostanoids such as PGF 2α , PGD 2 and PGI 2 (ref.43).

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-024-07254-x.

Mice
All mice used in this study were on a C57BL/6J genetic background and purchased from the Jackson Laboratory ( JAX).OT-I × CD45.1 mice were generated by crossing OT-I mice ( JAX, 003831) to CD45.1 ( JAX, 002014) mice.Ptger2 −/− Ptger4 fl/fl mice were generated by crossing Ptger2 −/− mice ( JAX, 004376) to Ptger4 fl/fl mice ( JAX, 028102) and further crossed to Cd4 cre mice ( JAX, 022071) to generate Cd4 cre Ptger2 −/− Ptger4 fl/fl mice or crossed to Gzmb cre mice ( JAX, 003734) to generate Gzmb cre Ptger2 −/− Ptger4 fl/fl mice.Unless stated otherwise, mice were on a CD45.2/CD45.2background.For some experiments, Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Ptger2 −/− Ptger4 fl/fl mice were crossed to OT-I mice to generate Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I mice and Ptger2 −/− Ptger4 fl/fl OT-I mice and used on a CD45.1/CD45.2 or CD45.1/CD45.1 background.WT or Rag1 −/− mice ( JAX, 002216) on a CD45.2/CD45.2 background were used as recipients in adoptive transfer experiments.In all experiments, mice at 6-12 weeks of age were sex-matched and randomly assigned to control or treatment groups.Mouse experiments with Ptgs1/Ptgs2 −/− BRAF V600E tumours and T cell depletion were conducted without blinding; all other experiments were performed in a blinded manner.No statistical methods were used to predetermine sample sizes.Mice were killed by cervical dislocation under anaesthesia.All mice were maintained and bred at the Klinikum rechts der Isar, TUM, or at the Klinikum der Universität München, LMU, under specific-pathogen-free, controlled conditions with a 12-h light-dark cycle, ambient temperature of 24 °C and humidity maintained at 55%, and in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations.All animal experiments were performed in accordance with the guidelines of the district government of upper Bavaria (Department 5-Environment, Health and Consumer Protection).

Cell lines
Control and Ptgs1/Ptgs2 −/− BRAF V600E melanoma cells were generated using the CRISPR-Cas9 system as previously described 14 .BRAF V600E -OVA and Ptgs1/Ptgs2 −/− BRAF V600E -OVA cells were generated by lentiviral transduction.In brief, OVA cDNA was subcloned into a pHIV-7 transfer vector carrying both the phosphoglycerate kinase (PGK) promoter and IRES-puromycin-resistance sequence.The production of third-generation self-inactivating lentiviral vectors, pseudotyped with VSV.G, was carried out as previously described 44 .Specifically, packaging cells were transfected and, after 2 days, cell supernatants were collected, filtered and used to transduce tumour cell lines in the presence of 8 µg ml -1 polybrene (Merck).After the incubation period, medium was exchanged for fresh medium, and target cells were passaged at least three times after transduction and selected using puromycin.MC38 cells were provided by A. Krüger, Institute of Experimental Oncology, TUM, and MC38-OVA and Panc02 cells were provided by V. Buchholz, Institute for Medical Microbiology, Immunology and Hygiene, TUM.

Tumour cell injections
Tumour cell lines were detached by trypsinization (Thermo Fisher Scientific) and washed three times in sterile PBS (Thermo Fisher Scientific).Unless stated otherwise, 2 × 10 6 cells were injected s.c. in 100 µl sterile PBS into the flank of each recipient mouse.Tumour growth was measured using a digital calliper.Tumour diameters stated in the figures refer to the average values of the longest diameter and its perpendicular for each tumour.A maximal tumour diameter of 15 mm served as the humane end point and was not exceeded in any of the experiments.

Processing of tumour tissue and lymphoid organs
Tumours, tdLNs or spleens of tumour-bearing mice were excised at the indicated time points after cell transplantation.Tumour or organ weight was determined using a microscale.For subsequent analyses by flow cytometry or cell sorting, tumour samples were mechanically dissociated and incubated with collagenase IV (200 U ml -1 , Thermo Fisher Scientific) and DNase I (100 µg ml -1 , Merck) for 40 min at 37 °C and filtered through a 70 µm and a 30 µm cell strainer (Miltenyi) to generate single-cell suspensions.Spleens were passed through a 70 µm cell strainer, followed by red blood cell lysis and a second filtration step using a 30 µm cell strainer.LNs were passed through a 30 µm cell strainer.For the isolation of migratory cDC1s, LNs were processed as described for tumour samples.

Antibodies and reagents for flow cytometry and cell sorting
The following antibodies and staining reagents were used for flow cytometry or cell sorting: fixable viability dye eFluor 450 (dilution: 1:500; Thermo Fisher Scientific); fixable viability dye APC-eF780

Flow cytometry and cell sorting
For staining of surface markers and viability dyes, cells were stained for 15 min at 4 °C in FACS buffer (PBS with 1% FCS and 2 mM EDTA).Staining of SIINFEKL-MHC class I complexes on cDC1s for analysis of OVA cross-presentation was performed for 40 min.For intracellular staining of GZMB, TCF1, Ki-67 and TOX, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) according to the manufacturer's protocol.Intracellular staining was performed overnight in permeabilization buffer at 4 °C.For intracellular staining of pSTAT5, cells were fixed and permeabilized using BD Cytofix (BD Biosciences) and BD Phosflow Perm Buffer III (BD Biosciences) according to the manufacturer's instructions (protocols II and III, BD Biosciences).For the detection of EdU incorporation, EdU was added to the culture at a final concentration of 15 µM for the last 3 h of the experiment, and analysis was performed using an EdU Proliferation kit (iFluor 488, Abcam) according to the manufacturer's protocol.
Generation of repetitively activated antigen-experienced TCF1 + CD8 + T cells TCF1 + CD8 + T cells were differentiated from splenic naive CD8 + T cells by repetitive activation as previously described 35 , with minor modifications.In brief, 1 × 10 6 naive CD8 + T cells were seeded in complete RPMI medium supplemented with 1× MEM non-essential amino acids solution and 1 mM sodium pyruvate.Low-dose IL-2 (85 U ml -1 ) and mouse anti-CD3/CD28 microbeads were added to the culture while maintaining a CD8 + T cell concentration of 1 × 10 6 cells per ml for multiple (re-) activation cycles over a course of 4 days, followed by purification of live cells by gradient centrifugation (Pancoll).

Analysis of IL-2Rγc expression and IL-2 signalling
TCF1 + CD8 + T cells from in vitro cultures were rested for 20 h in complete RPMI supplemented with low-dose IL-2 and purified by gradient centrifugation.Cells were stimulated with mouse anti-CD3/CD28 microbeads and low-dose IL-2 for 24 h in the absence or presence of PGE 2 (100 ng ml -1 ).After 24 h, IL-2Rγc chain expression was analysed by flow cytometry.For analysis of IL-2-induced STAT5 signalling, anti-CD3/ CD28 microbeads were removed by magnetic separation, cells were rested for 30 min at 37 °C in complete RPMI and stimulated for 30 min with different concentrations of IL-2 (10-100 U ml -1 , as indicated).After the incubation period, fixation buffer was directly added to the culture to terminate the signalling process and cells were stained for flow cytometry analysis.

PGE 2 measurements
Tumours and organs of tumour-bearing mice were excised 11 days after tumour cell transplantation, directly frozen in liquid nitrogen and stored at −80 °C until further processing.Samples were homogenized in homogenization buffer (0.1 M PBS, 1 mM EDTA and 10 µM indomethacin (Merck), pH 7.4) using a gentleMACS Dissociator (Miltenyi) followed by a freeze-thaw cycle.PGE 2 concentrations were measured by ELISA (Cayman Chemical) according to the manufacturer's protocol.

RNA isolation and quantitative real-time PCR
RNA was isolated using an Arcturus PicoPure RNA isolation kit (Thermo Fisher Scientific) and cDNA was generated using a SensiFAST cDNA synthesis kit (Bioline).Quantitative real-time PCR was carried out on a LightCycler 480 (Roche, LightCycler 480 software v.1.5.1) using a TAKYON No ROX SYBR MasterMix dTTP Blue kit (Eurogentec) according to the manufacturer's protocol.Ptger4 expression was determined using the ΔCt method, with Hprt serving as reference gene.Primer sequences were from a previous study 38 .All primers were purchased from Eurofins.
scRNA-seq and scTCR-seq CD8 + TILs were sorted from BRAF V600E tumours 11 days after tumour cell transplantation.A combination of cell hashing and DNA barcoding during library preparation was used for sample multiplexing, which enabled the simultaneous sequencing of four biological replicates from each group.For cell hashing, unique TotalSeq-C anti-mouse hashtag antibodies were used for hashing of cells from each experimental group as follows: WT: hashtag 1; Ptger2 −/− Ptger4 fl/fl : hashtag 2; Cd4 cre Ptger2 −/− Ptger4 fl/fl : hashtag 3; and Gzmb cre Ptger2 −/− Ptger4 fl/fl : hashtag 4 (1:250 each, Biolegend).Hashtagged cells from one tumour-bearing mouse of each group were pooled and loaded on a Chromium Next GEM Chip (10x Genomics).RNA-seq libraries were generated using Chromium Next GEM Single Cell 5′ Reagent kits v.2 User Guide with Feature Barcode technology for Cell Surface Protein (Rev D) according to the manufacturer's protocol (10x Genomics).Quality control was carried out using a High Sensitivity DNA kit (Agilent), a Bioanalyzer 2100 and a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific).For sequencing, libraries were pooled and analysed by paired-end sequencing (2 × 150 bp) on a NovaSeq6000 platform using S4 v.1.5(300 cycles) sequencing kits (Illumina).Libraries were sequenced to a depth of at least 2 × 10 4 reads per cell for gene expression libraries and 5 × 10 3 reads per cell for T cell receptor libraries.
Initial scRNA-seq analyses were performed for all samples from the groups Ptger2 −/− Ptger4 fl/fl , Cd4 cre Ptger2 −/− Ptger4 fl/fl and Gzmb cre Ptger2 −/− Ptger4 fl/fl , with data from the WT group being added at a later stage for validation of Ptger2 and Ptger4 read coverage (see below).Alignment of gene expression libraries and demultiplexing were performed using cellranger multi (Cell Ranger (v.6.1.1) 49; 10x Genomics) against the pre-built mouse reference v2020-A (10x Genomics, mm10/GRCm38, annotation from GENCODE Release M23) with the number of expected cells equals 21.000 as input argument.The BAM files were converted to FASTQ files using the tool bamtofastq with the argument --reads-per-fastq set to the total number of reads in the BAM file plus 10,000.After that, gene expression and TCR analysis were combined by running cellranger multi separately for each demultiplexed sample, disabling library concordance reinforcement.The algorithm was forced to find the number of cells identified in the first step of demultiplexing, and sample-specific FASTQ files were used as input for the gene expression analysis pipeline.The pre-built Ensembl GRCm38 Mouse V(D)J Reference v.5.0.0 was used for TCR analysis.
The initial downstream analysis was performed in R (v.4.0.4) with the R package Seurat (v.4.0.1) 50.Only cells with more than 1,000 genes detected, less than 10% of mitochondrial genes and with UMI counts less than 3 standard deviations above the mean were kept.The data were filtered for genes detected in at least three cells in one of the samples.Filtered read counts from each sample were normalized independently using sctransform (v.0.3.2) 51 with the glmGamPoi method 52 .Anchors between cells from different replicates were identified on the top 1,000 highly variable genes using canonical correlation analysis and 30 canonical vectors.Data integration was performed on first 20 PC analysis (PCA) dimensions.PCA was calculated for the integrated data on the top 1,000 highly variable genes and both k-nearest neighbour graph and UMAP were computed on the 30 nearest neighbours and first 20 PCA dimensions.Louvain clusters were identified using the shared nearest neighbour modularity optimization-based algorithm at resolutions 0.9, 0.65 and 0.9 for the groups Ptger2 −/− Ptger4 fl/fl , Cd4 cre Ptger2 −/− Ptger4 fl/fl and Gzmb cre Ptger2 −/− Ptger4 fl/fl , respectively.Contaminating myeloid cells were identified based on the average cluster expression of the marker genes Cd14, Lyz2, Fcgr3, Ms4a7, Fcer1g, Cst3, H2-Aa, Ly6d, Ms4a1 and Ly6d.Cycling cells were identified based on expression of Cdk1, Mcm2, Pclaf, H2afz, Birc5 and Mki67.
The integrative analysis between groups was performed in R (v.4.2.1) with the R package Seurat (v.4.1.1) 50.After general data pre-processing and regression of contaminating cells as mentioned above, filtered read counts from each sample were normalized independently using sctransform (v.0.3.2) 51 with glmGamPoi method 52 .Anchors between cells from all groups and all their replicates were identified using a more conservative approach, which led to weaker batch correction.For that purpose, reciprocal PCA was applied on the top 1,000 highly variable genes of each sample and anchors were picked using the first 20 dimensions and 1 neighbour only.PCA was performed on the integrated data on the top 1,000 highly variable genes.A k-nearest neighbour graph and UMAP (spread of 0.4, minimum distance of 0.01) were computed on the first 20 PCs and 30 nearest neighbours.A resolution of 0.6 was used for Louvain clusters identification using the shared nearest neighbour modularity optimization-based algorithm.DEGs between two groups were identified using the Wilcoxon rank-sum test and Bonferroni correction.Gene set expression scores at single-cell level were calculated using the AddModuleScore function, including only the detected genes.Similarity scores with reference datasets were calculated using the R package SingleR (v.1.10.0) 53with the top 200 DEGs.The processed transcriptome profiles of naive CD8 + T cells, memory stem cell CD8 + T cells and central memory CD8 + T cells were from a previous study 54 .For tumour antigen-specific CD8 + T cells in tdLNs, tumour-infiltrating stem-like CD8 + T cells and their naive counterparts, data from a previous study 3 were processed using the R package DESeq2 (v.1.36) 55.Gene set expression scores at the single-cell level were calculated using the AddModuleScore function, including only the detected genes.The effector T cell gene signature was from a previous study 56 (M3013: KAECH_NAIVE_VS_DAY8_EFF_CD8_TCELL_DN).The CD8 + T cell proliferation signature was obtained from MSigDB (GO:2000566).Transcriptional trajectories were inferred using the R package slingshot (v.2.4.0) 57 over the UMAP calculated on the integrated data, approximating the curves by 150 points.The pseudotime was calculated as a weighted average across lineages, weighted by the assignment weight.
TCR analysis of clonotype was performed using the R package scRepertoire (v.1.6.0) 58.Clonotypes were called based on a combination of VDJC genes comprising the TCR and the nucleotide sequence of the CDR3 region.Whenever the clonotype distribution is shown for individual groups, the cell number was downsampled, so that cluster 1 from all groups had the same maximum size.TF activity was inferred using the weighted mean method of decoupleR (v.2.2.2) 59 and TF-target interactions available through dorothea (v.1.8.0) 60 , with confidence levels A to C. Normalization to Ptger2 −/− Ptger4 fl/fl was achieved by subtracting its scores from the scores of the other groups.The top 100 variable TFs between clusters within each group were used to draw a network graph with tidygraph (v.1.2.1) 61 based on common targets with same defined mode of regulation as defined on the database.Only TFs with at least two common targets were kept for visualization.Louvain clusters were identified using igraph (v.1.3.2) 62 at a resolution of 0.5.
For addition of scRNA-seq data from the WT group, samples were pre-processed as described above and mapped to a reference formed by the integrated data of the Ptger2 −/− Ptger4 fl/fl , Cd4 cre Ptger2 −/− Ptger4 fl/fl and Gzmb cre Ptger2 −/− Ptger4 fl/fl groups using the R package Seurat (v.4.1.1) 50.For that purpose, anchors between cells from the reference and the WT groups along with all replicates were identified using reciprocal PCA on top 1,000 highly variable genes.Anchors were picked using the first 20 dimensions and 1 neighbour only.Annotations were transferred using the function TransferData, and data were integrated using Inte-grateEmbeddings. Cells from the added group were then projected onto the coordinates of the reference UMAP calling ProjectUMAP with 30 nearest neighbours.Read coverage was estimated using deepTools (v.3.5.4) 63 with bamCoverage and a bin size of 10 bp and normalization by bins per million mapped reads.For coverage analysis on Tcf7/TCF1 + and Tcf7/TCF1 − clusters, BAM files were split by cell barcodes from clusters 1-2 or clusters 3-8 using samtools (v.1.13) 64before coverage estimation.Read coverage on gene tracks was visualized using the R package trackViewer (v.1.32.1) 65 .

RNA-seq
In vitro generated, repetitively activated TCF1 + CD8 + T cells were incubated in the presence or absence of PGE 2 (100 ng ml -1 ) for 1 h at 37 °C followed by stimulation with IL-2 or IL-2 plus mouse anti-CD3/CD28 microbeads for an additional 4 h.Total RNA was isolated using Total RNA Miniprep (Monarch).Library preparation was carried out using a NEB Next UltraRNA Library Prep kit with i7 and i5 index reads of 8 bp each for mRNA library preparation and poly A enrichment.Sequencing was performed on a NovaSeq6000 PE150 platform in paired-end mode (read 1: 151 bp, read 2: 151 bp), using S4 (v.1.5)(300 cycles) sequencing kits (Illumina).Reads were aligned to the mouse reference genome (GRCm38/mm10, NCBI) using the Hisat2 (v.2.0.5) mapping tool.To quantify gene expression levels, featureCounts (v.1.5.0-p3) was used to count the reads mapped to each gene, followed by the calculation of fragments per kilobase of transcript sequence per million mapped reads based on gene length and read count.DEGs were identified using the DESeq2 R package (v.1.20.0).Adjusted P values were obtained using Wald test with multiple testing by the Benjamini-Hochberg method, and genes identified by DESeq2 with adjusted P values < 0.05 and fold change ≥ 2 were assigned as DEGs.Volcano plots were visualized using the ggplot2 R package ggplot2 (v.3.4.2), and PCA was conducted using the prcomp function in R and visualized using the R packages ggplot2 and ggrepel (v.0.9.3).DEGs obtained from comparing the groups 'anti-CD3/CD28 +IL-2' and 'PGE 2 -treated + anti-CD3/ CD28 +IL-2' were ordered based on their log 2 fold change values and subjected to GSEA using GSEA (v.4.3.2) probing for hallmark genes from mh.all.v2023.1.Mm (MSigDB).The PreRanked tool from GSEA (v.4.3.2) was used to determine the NES and significance by adjusted P values.

Statistical analyses
The GraphPad Prism software (v.9.5.0 and v.9.5.1) was used for statistical analyses.Affinity Designer (v.1.10.6)(Serif) was used to visualize data.Paired or unpaired two-tailed Student's t-test, one-way ANOVA or two-way ANOVA was used to assess statistical significance, as indicated in in the figure legends.Data are shown as the mean ± s.d., mean ± s.e.m. or box and whiskers plots, as indicated in the figure legends.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.e,f, Correlation of TCF1 + CD8 + TIL cluster gene expression with gene signatures of e naive CD8 + T cells, memory stem cell CD8 + T cells (TSCM) and central memory CD8 + T cells (TCM) or f naive CD8 + T cells, tumour antigen-specific CD8 + T cells in tdLNs and tumour-infiltrating stem-like CD8 + T cells.g, UMAP visualisation of transcript expression of indicated immune genes as determined by scRNA-seq.h, Expression levels of selected immune genes across CD8 + TIL clusters.i, Flow cytometric analysis of GZMB and CD62L expression among TCF1 + and TIM-3 + CD8 + TIL populations from a WT mouse.j, Flow cytometric analysis of TIM-3 and CXCR6 protein expression in CD8 + TILs from a WT mouse.k, Analysis of GZMB expression in activated (CD44 + ) CD8 + T cells isolated from tumours, tdLNs and spleen.Numbers indicate percentage of GZMB + cells compared to isotype control.Plots in a,c,i-k show data for one tumour representative for n = 6 tumours from one (a,b), two (c,i,k), or three ( j) independent experiments or pooled data from n = 4 biological replicates from one experiment (d-h).P values in e,f are from pairwise comparisons using Wilcoxon rank sum test and Bonferroni correction for multiple testing.P ≥ 0.05, not significant (NS).OT-I TILs.j, Analysis of TCF1 expression in Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I T cells in tdLNs and tumours over time.k, Experimental design for l. l, Effect of FTY720 treatment from day 6 on OT-I T cell expansion in tumours (n = 6).m,n, WT mice received 1 × 10 3 naive OT-I T cells or 1 × 10 3 naive Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I T cells i.v. and were transplanted s.c. with 2 × 10 6 D4M.3A-pOVA melanoma cells before m, quantification of OT-I cell expansion in tumours at different time points (n = 6) or n, analysis of tumour growth over time (n = 4).Data in b,h,l-n are pooled from two (h,l-n) or three (b) independent experiments and depicted as box plots extending from the 25 th to 75 th percentiles with the median as centre and whiskers corresponding to minimum and maximum values (b,h,l,m) or shown as mean ± s.e.m. (n).Plots in c,d,g,i,j show data for one sample representative for (c,d,g,i) n = 7 or ( j) n = 6 samples from two independent experiments.P values in b,l are from one-way ANOVA with Tukey's multiplecomparison test, P values in h,m are from unpaired t-test, P-values in n are from two-way ANOVA with Bonferroni's correction for multiple testing.not significant (NS).

Fig. 2 |
Fig. 2 | Ablation of T cell-intrinsic EP 2 /EP 4 signalling rescues CD8 + T cell expansion and effector differentiation in PGE 2 -producing tumours.a-g, scRNA-seq analyses of CD8 + TILs in control BRAF V600E tumours from Ptger2 −/− Ptger4 fl/fl mice, Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb ce Ptger2 −/− Ptger4 fl/fl mice (n = 4 each).a, Experimental design.b, Uniform manifold approximation and projection (UMAP) plot of 12,516 CD8 + TILs coloured according to cluster classification.c, Visualization of Tcf7 and Havcr2 transcript levels.d, PCPT plot showing expression levels of selected genes.e, Developmental trajectory prediction by unsupervised slingshot analysis.f,g, Comparison of CD8 + TIL clusters among Ptger2 −/− Ptger4 fl/fl mice, Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice.f, Density analysis.g, Quantification relative to cluster 1 (n = 4 each).h,i, scTCR-seq analyses of CD8 + TILs from n = 3 tumours for each group.h, UMAP visualizations of T cell clonotype distribution.i, Quantification of T cell clonotype frequency.j-n, TIM-3 + effector CD8 + T cell differentiation in tumour tissue.Cd4 cre Ptger2 −/− Ptger4 fl/fl mice bearing control BRAF V600E tumours were injected with FTY720 or NaCl as control.j, Experimental design.k, Representative flow cytometry plots showing TCF1 and TIM-3 expression among CD44 + CD8 + TILs.l, Average percentages of CD8 + TIL populations across n = 6 tumours.m, Quantification of CD8 + TIL numbers (n = 6).n, Analysis of tumour mass (n = 10).Anti-CD8β, antibody-mediated CD8 + T cell depletion in the absence of FTY720 treatment.Data in a-h are from one experiment.Data in g are depicted as box plots extending from the 25th to 75th percentiles with the median as the centre and the whiskers corresponding to the minimum and maximum values.Data in k-n are pooled from two (k,l,m) or three (n) independent experiments and depicted as the mean ± s.e.m.P values are from two-way ANOVA with Bonferroni's correction for multiple testing (g) or one-way ANOVA with Tukey's multiple-comparison test (m,n).Plots in k show data for 1 tumour representative for n = 6 tumours from 2 independent experiments.

6 PFig. 3 |
Fig. 3 | PGE 2 impairs CD8 + T cell expansion and effector differentiation from TCF1 + cells by inhibiting IL-2 signalling.a, TF activity in TCF1 + CD8 + TILs from control BRAF V600E tumours in Cd4 cre Ptger2 −/− Ptger4 fl/fl mice and Gzmb cre Ptger2 −/− Ptger4 fl/fl mice (relative to Ptger2 −/− Ptger4 fl/fl mice).b, Effect of PGE 2 on ex vivo expansion of TCF1 + CD8 + TILs sorted from Ptgs1/Ptgs2 −/− BRAF V600E tumours (n = 3).c,d, Effect of PGE 2 on expansion (c) and proliferation (d) of repetitively activated TCF1 + CD8 + T cells from in vitro T cell cultures (n = 4).e-h, Analysis of repetitively activated TCF1 + CD8 + T cells by RNA-seq (n = 4).e, Experimental design.f, principal component (PC) analysis based on all DEGs.g, Volcano plot showing the effect of PGE 2 exposure on gene expression in TCF1 + CD8 + T cells stimulated with anti-CD3/CD28 and IL-2.h, GSEA of hallmark pathways based on g. *Pathways significantly regulated; NES, normalized enrichment score.i,j, Effect of PGE 2 exposure on IL-2-dependent pSTAT5 induction in repetitively activated TCF1 + CD8 + T cells.Cells were treated with 33 U ml -1 IL-2.j, n = 3. k, Expansion of repetitively activated TCF1 + CD8 + T cells treated or untreated with PGE 2 and stimulated as indicated (n = 3).l-n, WT or Cd4 cre Ptger2 −/− Ptger4 fl/fl TCF1 + CD8 + T cells from in vitro T cell cultures were incubated with or without PGE 2 for 20 h before stimulation with IL-2 (l,m) or anti-CD3/CD28 and IL-2 (n).l, Flow cytometry plot showing pSTAT5 signalling after 30 min.Cells were treated with 33 U ml -1 IL-2.m, Quantification of pSTAT5 (n = 3).n, Quantification of T cell expansion (n = 3).Data in b and c are pooled from two independent experiments.Data in j, k, m and n are representative of two independent experiments.Plots in d, i and l show data for 1 T cell culture representative of n = 6 T cell cultures analysed in 2 independent experiments.For b, c, k and n, horizontal lines and error bars indicate the mean ± s.e.m.For j and m, box plots indicate the median.P values are from unpaired t-tests.In g, DEGs (P < 0.05; fold change ≥ 2) were identified by Wald test with multiple testing using the Benjamini-Hochberg method.

3 Fig. 4 |
Fig. 4 | EP 2 /EP 4 -deficient tumour antigen-specific CD8 + T cells expand in PGE 2 -producing tumours and mediate tumour immune control.a, Experimental design for b-f.b, Flow cytometric plots of CD8 + T cells from tdLNs and tumours from the indicated days.c,d, Numbers of expanded OT-I CD8 + T cells in tdLNs (c) and tumours (d) at indicated time points (n = 6).e,f, Analysis of CD44 and CXCR6 expression in Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I cells.e, Flow cytometry plots.f, Subset frequencies (n = 6).g-j, Effect of CD122/ CD132 blockade on OT-I T cell expansion in tumours.g-j, Effect of anti-CD122 and anti-CD132 (anti-CD122/CD132) treatment on OT-I TIL expansion in WT mice with control or Ptgs1/Ptgs2 −/− BRAF V600E -OVA tumours or with MC38-OVA tumours, analysed 11 days after tumour transplantation.g,h, Flow cytometry plots (g) and OT-I TIL numbers (h) in BRAF V600E -OVA tumours (n = 6).i,j, Flow cytometry plots (i) and OT-I TIL numbers ( j) in MC38-OVA tumours (n = 10).k, Experimental design for l and m with MC38-OVA tumours.l, Flow cytometry plot (left) and quantification (right) of OT-I TILs at day 10 (n = 6).m, Flow cytometry plots showing the population size of TIM-3 + CXCR6 + cells among control and Cd122 −/− Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I TILs.n, WT mice received 1 × 10 3 naive OT-I T cells or 1 × 10 3 naive Cd4 cre Ptger2 −/− Ptger4 fl/fl OT-I T cells intravenously (i.v.) and were transplanted s.c. with 2 × 10 6 MC38-OVA cells before analysis of tumour growth over time (n = 10).Asterisk indicates that termination criteria were reached.Data in c, d, f, h, j, l and n are pooled from two (c,d,h,l) or three (f,j,n) independent experiments and depicted as box plots extending from the 25th to 75th percentiles with the median as the centre and the whiskers corresponding to minimum and maximum values (c,d,h,j,l) or shown as the mean ± s.e.m. (f,n).Plots in b, e, g, i, l and m show data for 1 sample representative of n = 6 samples analysed in 2 (b,g,l,m) or 3 (e,i) independent experiments.P values are from paired t-tests (l), one-way ANOVA with Tukey's multiple-comparison test (c,d) or Dunnett's multiple-comparison test (h,j), or two-way ANOVA with Bonferroni's correction for multiple testing (n).